I am doing double immunostaining and first primary Ab is raised in GOAT (Polyclonal goat IgG against Human CD80) and second primary AB is raised in MOUSE(Monoclonal Anti-Vinculin). For the First primary AB I have chosen Alexa flour-568 Rabbit-Anti-Goat IgG (A-11070, Invitrogen) as secondary AB and for the second primary Ab, can I choose "Alexa flour-488-Goat Anti-Mouse, IgG (A-10680, Invitrogen).
Note: The first primary AB and secondary AB for the second primary AB is raised in GOAT??.
Question: whether the both antibodies will bind to Alexa fluor 568?
In my opinion it is just a matter of the right sequence. The Rabbit-anti-Goat has to be first applied and later on the Goat-anti-Mouse. if the other way round, the Rabbit-anti-Goat will certainly bind onto the Goat-anti-Mouse. Simultan apllication is not possible. But I am with Jennifer, that her recommended variant provides less problems.
If I've read correctly, you want to double stain using a 568 rabbit anti-goat secondary and a 488 goat anti-mouse secondary. This isn't ideal because the anti-goat 568 will recognize the 488 antibody raised in goat.
When I did something similar by mistake, I got perfect co-localization of the 568 to the 488 expression pattern for non-overlapping proteins, suggesting that it was due to the secondary antibody clash.
I would suggest using the 568 rabbit anti-goat with a 488 rabbit anti-mouse, which should be easy to find.
In my opinion it is just a matter of the right sequence. The Rabbit-anti-Goat has to be first applied and later on the Goat-anti-Mouse. if the other way round, the Rabbit-anti-Goat will certainly bind onto the Goat-anti-Mouse. Simultan apllication is not possible. But I am with Jennifer, that her recommended variant provides less problems.
1. The species differences between mouse and rabbit isn't large enough. So the suggestion to use a direct labeling is a good one.
2. Try every antibody allone. About 50% of all (murine) monoclonals are not only cross reactive, they are multireactive. Multireactive = antigen recognition without the presence of any relationship (AA sequence, etc.). Check them on a panel of different antigens, compare reaction patterns of antibodies recognizing the same antigen, compare different methods presentig the same antigen (e.i. western blots, immunohistolog with other organs from other species, ...)
Thanks for your answers. I have tried as suggested by Gudrun Lang/Goodwin Jinesh. I am able to get proper localisation and also nonspecific binding. I need to standridise one of the primary antibody and blocking solution.
Blocking solution: I used 1%BSA in PBST. I have not used rabbit/goant normal serum.
Which blocking solution is good or do I have to use secondary antibody from one species?
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